Technologies, Methods, and Products of Small Molecule Directed Tissue and Organ Regeneration from Human Pluripotent Stem Cells

ABSTRACT

Pluripotent human embryonic stem cells (hESCs) hold great potential for restoring tissue and organ function, which has been hindered by inefficiency and instability of generating desired cell types through multi-lineage differentiation. This instant invention is based on the discovery that pluripotent hESCs maintained under defined culture conditions can be uniformly converted into a specific lineage by small molecule induction. Retinoic acid induces specification of neuroectoderm direct from the pluripotent state of hESCs and triggers progression to neuronal progenitors and neurons efficiently. Similarly, nicotinamide induces specification of cardiomesoderm direct from the pluripotent state of hESCs and triggers progression to cardiac precursors and cardiomyocytes efficiently. This technology provides a large supply of clinically-suitable human neuronal or cardiac therapeutic products for CNS or myocardium repair. This invention enables well-controlled efficient induction of pluripotent hESCs exclusively to a specific clinically-relevant lineage for tissue and organ engineering and regeneration, cell-based therapy, and drug discovery.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/306,114 filed onNov. 29, 2011, which issued on May 6, 2014 as U.S. Pat. No. 8,716,017,and claims priority to U.S. provisional patent application Ser. No.61/458,965 filed on Dec. 6, 2010.

The priority application is hereby incorporated herein by reference inits entirety.

GOVERNMENT INTEREST

This invention was made with government support under Grant No. AG024496and HD056530 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

SPECIFICATION

Inventors: Parsons, Xuejun Huang (San Diego, CA) Correspondence Xuejun HParsons, Name and 4539 Donald Ave, San Diego, CA 92117, USA Address andCustomer number: 000101791 Customer#: Assignee: Xcelthera, Inc. Filed:Oct. 22, 2014 Current U.S. 435/1.1; 435/1.2; 435/1.3; 435/4; 435/366;435/368; Class: 435/374; 435/377; 435/404; 435/405 International C12N5/00; C12N 5/02; C12N 5/071; C12N 5/073; Class: C12N 5/0735; C12N5/0789; C12N 5/079; C12N 5/0793; C12N 5/0797; C12N 5/095; C12N 5/22;A61P 9/00; A61P 9/04; A61P 9/10; A61P 25/00; A61P 25/16; A61P 25/28Field of Search: 435/1.1, 1.2, 1.3, 4, 366, 368, 374, 377, 404, 405

REFERENCES CITED

-   1. Parsons X H, Teng Y D, Moore D A, Snyder E Y. (2011) Patents on    technologies of human tissue and organ regeneration from pluripotent    human embryonic stem cells. Recent Patents on Regenerative Medicine    1:142-163. PMID: 2335596. PMCID: 3554241.-   2. Parsons X H. (2013) Embedding the future of regenerative medicine    into the open epigenomic landscape of pluripotent human embryonic    stem cells. Ann. Rev. Res. Biol. 3(4):323-349. PMID: 25309947.    PMCID: 4190676.-   3. Redmond D E Jr, et al. (2007) Behavioral improvement in a primate    Parkinson's model is associated with multiple homeostatic effects of    human neural stem cells. Proc Natl Acad Sci USA 104:12175-12180.    PMID: 17586681. PMCID: 1896134.-   4. Parsons X H, Teng Y D, Parsons J F, Snyder E Y, Smotrich D B,    Moore D A. (2011) Efficient derivation of human cardiac precursors    and cardiomyocytes from pluripotent human embryonic stem cells with    small molecule induction. JoVE 57:e3274. DOI: 10.3791/3274.    PMID: 22083019. PMCID: 3308594.-   5. Parsons J F, Smotrich D B, Gonzalez R, Snyder E Y, Moore D A,    Parsons X H. (2012) Defining conditions for sustaining epiblast    pluripotence enables direct induction of clinically-suitable human    myocardial grafts from biologics-free hESCs. J. Clinic. Exp.    Cardiology S9:001. DOI: 10.4172/2155-9880.S9-001. PMID: 22905333.    PMCID: 3419496.-   6. Parsons X H. (2012) The dynamics of global chromatin remodeling    are pivotal for tracking the normal pluripotency of human embryonic    stem cells. Anatom. Physiol. S3:002. DOI: 10.4172/2161-0940.S3-002.    PMID: 23543848. PMCID: 3609651.-   7. Parsons X H, Teng Y D, Parsons J F, Snyder E Y, Smotrich D B,    Moore D A. (2011) Efficient derivation of human neuronal progenitors    and neurons from pluripotent human embryonic stem cells with small    molecule induction. JoVE 56:e3273. DOI: 10.3791/3273.    PMID: 22064669. PMCID: 3227216.-   8. Parsons X H. (2012) MicroRNA profiling reveals distinct    mechanisms governing cardiac and neural lineage-specification of    pluripotent human embryonic stem cells. J. Stem Cell Res. Ther.    2:124. DOI: 10.4172/2157-7633.1000124. PMID: 23355957. PMCID:    3554249.-   9. Parsons X H. (2012) An engraftable human embryonic stem cell    neuronal lineage-specific derivative retains embryonic chromatin    plasticity for scale-up CNS regeneration. J. Reg. Med. & Tissue Eng.    1:3. DOI: 10.7243/2050-1218-1-3. PMID: 23542901. PMCID: 3609668.-   10. Parsons X H, Parsons J F, Moore D A. (2013) Genome-scale mapping    of microRNA signatures in human embryonic stem cell neurogenesis.    Mol. Med. Ther. 1:2. DOI: 10.4172/2324-8769.1000105. PMID: 23543894.    PMCID: 3609664.-   11. Parsons X H. (2013) Human stem cell derivatives retain more open    epigenomic landscape when derived from pluripotent cells than from    tissues. J. Regen. Med. 1:2. DOI: 10.4172/2325-9620.1000103.    PMID: 23936871. PMCID: 3736349.-   12. Parsons X H. (2013) Constraining the pluripotent fate of human    embryonic stem cells for tissue engineering and cell therapy—the    turning point of cell-based regenerative medicine. British    Biotech. J. 3(4):424-457. PMID: 24926434. PMCID: 4051304.-   13. Parsons X H. (2014) Direct conversion of pluripotent human    embryonic stem cells (human E S cells) under defined culture    conditions into human neuronal or cardiomyocytes cell therapy    derivatives. Methods Mol. Biol. 2014 Feb. 6. Chapter in Human    Embryonic Stem Cells: Methods and Protocols, 2^(nd) Edition.    Springer's Protocols. DOI: 10.1007/7651_2014_69 PMID: 24500898.

INCORPORATION BY REFERENCE

This application is a divisional of U.S. Ser. No. 13/306,114 filed onNov. 29, 2011, which issued on May 6, 2014 as U.S. Pat. No. 8,716,017,and claims benefit of and priority to U.S. provisional patentapplication Ser. No. 61/458,965 filed on Dec. 6, 2010, which is herebyincorporated by reference as if fully set forth.

FIELD OF THE INVENTION

The present invention relates generally to the fields of human embryonicstem cell biology and regenerative medicine. Specifically, thisinvention provides technologies, methods and products forwell-controlled efficient direct induction of human pluripotent stemcells exclusively to a specific neural or cardiac lineage using smallmolecules for use in research, drug screening, tissue and organengineering, tissue and organ regeneration, cell-based therapy, andclinics.

BACKGROUND OF THE INVENTION

Human embryonic stem cells (hESCs) have the unconstrained capacity forlong-term stable undifferentiated growth in culture and the intrinsicpotential for differentiation into all somatic cell types in the humanbody [1, 2]. Derivation of hESCs, essentially the in vitrorepresentation of the pluripotent inner cell mass (ICM) or epiblast ofthe human blastocyst, provides not only a powerful in vitro model systemfor understanding the human embryonic development, but also apluripotent reservoir for in vitro derivation of a large supply ofdisease-targeted human somatic cells that are restricted to the lineagein need of repair [1, 2]. However, how to channel the widedifferentiation potential of human pluripotent cells efficiently andpredictably to a desired phenotype has been a major challenge for bothdevelopmental study and clinical translation. Conventional approachesrely on multi-lineage inclination of pluripotent cells throughspontaneous germ layer differentiation, which yields mixed populationsof cell types that may reside in three embryonic germ layers and oftenmakes desired differentiation not only inefficient, but uncontrollableand unreliable as well [1, 2]. Although such cells can differentiatespontaneously in vitro into cells of all germ layers by going through amulti-lineage aggregate or embryoid body stage, only a small fraction ofcells pursue a given lineage. In those hESC-derived multi-lineageaggregates or embryoid bodies, the simultaneous appearance of asubstantial amount of widely divergent undesired cell types that mayreside in three embryonic germ layers often makes the emergence ofdesired phenotypes not only inefficient, but uncontrollable andunreliable as well. Following transplantation, thesepluripotent-cell-derived grafts tend to display not only a lowefficiency in generating the desired cell types necessary forreconstruction of the damaged structure, but also phenotypicheterogeneity and instability, hence, a high risk of tumorigenicity [1,2]. Currently, the first-generation of hESC-derived cellular productscontains variable levels of mixed populations of cell types, includingresidual undifferentiated hESCs and partially differentiated cells thatretain the capacity to proliferate and differentiate into unwantedcells, raising a potential safety concern. In view of growing interestin the use of human pluripotent cells, includingartificially-reprogrammed human induced pluripotent stem cells (hiPScells) from non-embryonic or adult cell sources, teratoma formation andthe emergence of inappropriate cell types have become a constant concernfollowing transplantation [1, 2]. Without a practical strategy toconvert pluripotent cells direct into a specific lineage, previousstudies and profiling of pluripotent hESCs and their differentiatingmulti-lineage aggregates have provided little implications to molecularcontrols in human embryonic development. Developing a novel practicalapproach that permits to channel the wide differentiation potential ofhuman pluripotent cells efficiently and predictably to a desiredphenotype is not only vital to harnessing the power of hESC biology forsafe and effective cell-based therapies, but also crucial for unveilingthe molecular and cellular cues that direct human embryogenesis.

The hESC lines initially were derived and maintained in co-culture withgrowth-arrested mouse embryonic fibroblasts (MEFs) [1]. Although severalhuman feeder, feeder-free, and chemically-formulated culture systemshave been developed for hESCs, the elements necessary and sufficient forsustaining the self-renewal of human pluripotent cells remain unsolved[1]. These exogenous feeder cells and biological reagents help maintainthe long-term stable growth of undifferentiated hESCs whereas mask theability of pluripotent cells to respond to developmental signals.Therefore, a defined culture system for maintenance of hESCs might notonly render specification of clinically-relevant early lineages directlyfrom the pluripotent state without an intervening multi-lineagegerm-layer or embryoid body stage, but also allow identify the signalingmolecules necessary and sufficient for inducing the cascade oforganogenesis in a process that may emulate the human embryonicdevelopment [1].

Current therapeutic approaches for a wide range of neurological diseasesand injuries provide symptomatic relief but none of them change theprognosis of disease. Therefore, there is a large unfulfilled need forcell-based therapies to provide regeneration and replacement options torestore the lost nerve tissue and function. However, to date, lacking ofa clinically-suitable source of engraftable human stem/progenitor cellswith adequate neurogenic potential has been the major setback indeveloping safe and effective cell-based therapies for restoring thedamaged or lost central nervous system (CNS) structure and circuitry ina wide range of neurological disorders. The traditional sources ofengraftable human stem cells with neural potential for transplantationtherapies have been multipotent human neural stem cells (hNSCs) isolateddirectly from the CNS [3]. These CNS-derived primary hNSCs areneuroepithelial-like cells that are positive for nestin and canspontaneously differentiate into a mixed population of cells containingundifferentiated hNSCs, neurons, astrocytes, and oligodendrocytes invitro and in vivo [3]. However, cell therapy based on CNS tissue-derivedhNSCs has encountered supply restriction and difficulty to use in theclinical setting due to their declining plasticity with aging andlimited expansion ability, which makes it difficult to maintain a largescale and prolonged culture and potentially restricts the tissue-derivedhNSC as an adequate source for graft material in the clinical setting[3]. Despite some beneficial outcomes, CNS-derived hNSCs appeared toexert their therapeutic effect primarily by their non-neuronal progeniesthrough producing trophic and/or neuroprotective molecules to rescueendogenous dying host neurons [1, 3]. The engrafted tissue-derivedstem/progenitor cells generated a small number of neurons that wereinsufficient to achieve the anticipated mechanism of neuron replacementin the damaged CNS [1, 3].

The genetically stable pluripotent hESCs proffer cures for a wide rangeof neurological disorders by supplying the diversity of human neuronalcell types in the developing CNS for regeneration and repair. Therefore,they have been regarded as an ideal source to provide an unlimitedsupply of human neuronal cell types and subtypes for restoring thedamaged or lost nerve tissue and function in CNS disorders. However,realizing the developmental and therapeutic potential of hESCs has beenhindered by the inefficiency and instability of generating desired celltypes from pluripotent cells through multi-lineage differentiation.Although neural lineages appear at a relatively early stage indifferentiation, <5% hESCs undergo spontaneous differentiation intoneurons [1]. Retinoic acid (RA) does not induce neuronal differentiationof undifferentiated hESCs maintained on feeders [1]. And unlike mouseESCs, treating hESC-differentiating multi-lineage aggregates—embryoidbodies (EBs)—only slightly increases the low yield of neurons [1, 2].Under protocols presently employed in the field, these neural graftsderived from pluripotent cells through multi-lineage differentiationyielded neurons at a low prevalence following engraftment, which werenot only insufficient for regeneration or reconstruction of the damagedCNS structure, but also accompanied by unacceptably high incidents ofteratoma and/or neoplasm formation [1]. Similar to CNS-derived hNSCs,these hESC-derived hNSCs are neuroepithelial-like cells that arepositive for nestin and can spontaneously differentiate into a mixedpopulation of cells containing undifferentiated hNSCs, neurons,astrocytes, and oligodendrocytes in vitro and in vivo [1, 2]. Beforefurther differentiation, those secondary hNSCs were mechanicallyisolated or enriched from hESC-differentiating multi-lineage aggregatesor embryoid bodies. Similar to their CNS counterpart, the therapeuticeffect of these hESC-derived hNSCs was mediated by neuroprotective ortrophic mechanism to rescue dying host neurons, but not related toregeneration from the graft or host remyelination [1, 2]. Growingevidences indicate that these secondary hNSCs derived from hESCs viaconventional multi-lineage differentiation in vitro appear to haveincreased risk of tumorigenicity but not improved neurogenic potentialcompared to primary hNSCs isolated from the CNS tissue in vivo,remaining insufficient for CNS regeneration [1, 2].

To date, the lack of a suitable human cardiac cell source has been themajor setback in regenerating the damaged human myocardium, either byendogenous cells or by cell-based transplantation or cardiac tissueengineering [1, 2]. In the adult heart, the mature contracting cardiacmuscle cells (cardiomyocytes) are terminally differentiated and unableto regenerate. Damaged or diseased cardiomyocytes are removed largely bymacrophages and replaced by non-functional cells or scar tissue.Although cell populations expressing stem/progenitor cell markers havebeen identified in postnatal hearts, the minuscule quantities andgrowing evidences indicating that they are not genuine heart cells andthat they differentiate predominately to smooth muscle cells rather thanfunctional contractile cardiomyocytes have caused skepticism if they canpotentially be harnessed for cardiac repair [1, 2]. There is no evidencethat stem/precursor/progenitor cells derived from other sources, such asmesenchymal stem cells, bone marrow cells, umbilical cord stem cells,cord blood cells, patients' heart tissue, placenta, or fat tissue areable to give rise to the contractile heart muscle cells followingtransplantation into the heart [1, 2]. Therefore, the need to regenerateor repair the damaged heart muscle (myocardium) has not been met byadult stem cell therapy, either endogenous or via cell delivery, intoday's healthcare industry. Pluripotent hESCs proffer unique revenue togenerate a large supply of cardiac lineage-committed cells as humanmyocardial grafts for cell-based therapy. Due to the prevalence ofcardiovascular disease worldwide and acute shortage of donor organs oradequate human myocardial grafts, there is intense interest indeveloping hESC-based therapy for heart disease and failure [1, 2]. ThehESCs and their derivatives are considerably less immunogenic than adulttissues [1, 2]. It is also possible to bank large numbers of humanleukocyte antigen isotyped hESC lines so as to improve the likelihood ofa close match [1, 2].

However, realizing the therapeutic potential of hESCs has been hinderedby the inefficiency and instability of generating cardiac cells frompluripotent cells through multi-lineage differentiation. InhESC-differentiating multi-lineage aggregates (embryoid body), only avery small fraction of cells (˜1-4%) spontaneously differentiate intocardiomyocytes [1, 2]. Following mechanical isolation andimmuno-selection, the small quantity of enriched cardiomyocytes couldrescue the function of a damaged myocardium as a biological pacemakerfollowing injection into the heart of animal models [1, 2]. Althoughsuch hESC-derived cardiomyocytes can attenuate the progression of heartfailure in rodent models of acute myocardial infraction, they areinsufficient to restore heart function or to alter adverse remodeling ofa chronic myocardial infarction model following transplantation [1, 2].

It can therefore be seen that there is a need to develop new techniquesfor well-controlled efficiently channeling the wide differentiationpotential of pluripotent hESCs exclusively and predictably to a largescale of neuronal lineage committed cells, which is vital to providing alarge supply of clinically-suitable human neuronal therapeutic productsacross the spectrum of developmental stages in high purity andefficiency, and with adequate neurogenic potential for neuronal repairagainst neurological diseases or injuries.

It can therefore be seen that there is a need to develop new techniquesfor well-controlled efficiently channeling the wide differentiationpotential of pluripotent hESCs exclusively and predictably to a largescale of cardiac lineage committed cells, which is vital to providing alarge supply of clinically-suitable human cardiac therapeutic productsacross the spectrum of developmental stages in high purity andefficiency, and with adequate cardiogenic potential for myocardiumrepair against cardiovascular diseases.

SUMMARY OF THE INVENTION

The present invention provides the techniques on direct conversion ofpluripotent human embryonic stem cells (hESCs) uniformly into a specificclinically-relevant lineage by small molecule induction.

The present invention provides the technique for efficient production ofhuman neuronal progenitors and human neuronal cell types and subtypes inthe developing CNS from pluripotent hESCs for neuronal regeneration andreplacement therapies against a wide range of neurological disorders.

The present invention provides the technique for efficient production ofhuman cardiac precursors and human cardiomyocytes from pluripotent hESCsfor myocardium regeneration and replacement therapies against heartdisease and failure.

Accordingly, one embodiment of the invention is provided a method ofidentifying conditions for well-controlled efficient induction ofpluripotent hESCs, maintained under a defined culture system that iscapable of insuring the proliferation of undifferentiated hESCs,exclusively to a specific clinically-relevant lineage by small moleculeinduction.

Another preferred embodiment of the invention is provided a method oflineage-specific differentiation of human pluripotent stem cells tospecialized functional cells by small molecule induction.

A particular embodiment of the invention is provided a method of usingretinoic acid (RA) to induce the specification of neuroectoderm directfrom the pluripotent state of hESCs in a defined culture platform bypromoting nuclear translocation of the neuronal-specific transcriptionfactor Nurr-1 and trigger the progression to human neuronal progenitorsand human neurons in high efficiency, purity, and neuronal lineagespecificity.

Another particular embodiment of the invention is provided a method ofusing nicotinamide (NAM) to induce the specification of cardiomesodermdirect from the pluripotent state of hESCs in a defined culture platformby promoting the expression of the earliest cardiac-specifictranscription factor Csx/Nkx2.5 and trigger the progression to cardiacprecursors and beating cardiomyocytes in high efficiency, purity, andcardiac lineage specificity.

These and other embodiments of the invention are further elucidated inthe description that follows.

DESCRIPTION OF THE DRAWINGS

DESCRIPTION OF THE FIGURES. FIGS. 1-11 represent data from theexperiments supporting the invention.

FIG. 1. (a) Overall scheme of the invention of lineage-specificdifferentiation of human pluripotent stem cells (hESCs) by smallmolecule induction for high efficient direct conversion of pluripotenthESCs uniformly into a specific clinically-relevant functional lineage.A schematic of well-controlled efficient specification of pluripotenthESCs directly and exclusively to a functional neural or cardiac lineageby small molecule induction.

(b) Comparison of neuronal differentiation efficiencies of pluripotenthESCs by our invention (NEURONAL: neuronal lineage-specificdifferentiation of pluripotent hESCs directly and exclusively to cellsof a neuronal lineage by small signal molecule induction) withconventional multi-lineage differentiation approaches throughspontaneous germ-layer induction of pluripotent cells to neural cells(CONTROL). Our novel technology shows a drastic increase in neuronaldifferentiation efficiency (>90% of cells expressing neuronal markers,e.g., beta-III-tubulin) when compared to similarly cultured cellsderived from embryoid bodies (<5% of cells expressing neuronal markers)by conventional approaches of multi-lineage differentiation ofpluripotent cells.

(c) Comparison of cardiac differentiation efficiencies of pluripotenthESCs by our invention (CARDIAC: cardiac lineage-specificdifferentiation of pluripotent hESCs directly and exclusively to cellsof a cardiac lineage by small signal molecule induction) withconventional multi-lineage differentiation approaches throughspontaneous germ-layer induction of pluripotent cells to cardiac cells(CONTROL). Our novel technology shows a drastic increase in cardiacdifferentiation efficiency (>90% of cells expressing cardiac markers,e.g., cardiac specific transcriptional factor Csx/Nkx2.5) when comparedto similarly cultured cells derived from embryoid bodies (<4% of cellsexpressing cardiac markers) by conventional approaches of multi-lineagedifferentiation of pluripotent cells.

FIG. 2. Small molecules signal cardiac or neural induction direct ofpluripotence under defined conditions.

(a) Upon exposure of undifferentiated hESCs to nicotinamide (NAM) orretinoic acid (RA) under the defined culture system, all the cellswithin the colony underwent morphology changes to large differentiatedcells that down-regulated (with NAM) or ceased (with RA) expressingpluripotence-associated markers, as indicated by Oct-4 (red).

(b) Cardiac fate switch direct of the pluripotent state of hESCs inducedby nicotinamide. NAM-induced Oct-4-negative cells began to express thecardiac specific transcription factor (Csx) Nkx2.5 (green) andalpha-actinin (red), consistent with early cardiac differentiation.Progressively increased intensity of Nkx2.5 was usually observed inareas of the colony where cells began to pile up. These differentiatedcells did not express markers for other lineages, including AFP (red),Pdxl (green) [endoderm], Map-2 (red), GFAP (green), HNK1 (red), and Pax6(green) [ectoderm]. All cells are indicated by DAPI staining of theirnuclei (blue). Insets at the top better visualize individual cells athigher-magnification. These data suggested that NAM was sufficient forinducing the pluripotent hESCs maintained in the defined culture systemto transition from a pluripotent state exclusively to a cardiomesodermalphenotype.

(c) Neural fate switch direct of the pluripotent state of hESCs inducedby retinoic acid. RA-induced differentiated Oct-4-negative cells beganto express HNK1 (red), AP2 (red), TrkC (green), and β-III-tubulin (red),consistent with early neuroectodermal differentiation, but not markersassociated with other lineages, including Pdxl (red), AFP (red), andinsulin (green) [endoderm], Nkx2.5 (green) [mesoderm], and GFAP (green)[glial cells]. These differentiated cells continued to multiply and thecolonies increased in size, proceeding spontaneously to matureultimately expressing the neuronal marker Map-2 (green), usually inareas where cells began to pile up. All cells are indicated by DAPIstaining of their nuclei (blue). Insets at the top better visualizeindividual cells at higher-magnification. These data suggested that RAwas rendered sufficient to induce hESCs maintained in the definedculture system to transition from a pluripotent state exclusively to aneuroectodermal phenotype.

FIG. 3. The cardiac- or neural-induced hESCs capable of progression tobeating cardiomyocytes or ventral neurons with high efficiency.

(a) The induced hESCs formed cardioblasts (Nkx2.5+, with NAM) orneuroblasts (β-III-tubulin+, with RA) in suspension, as compared togerm-layer-induced multi-lineage embryoid bodies (EBs) derived fromhESCs without treatment (Control) over the same time period.

(b) NAM-induced hESCs yielded beating cardiomyocytes with a drasticincrease in efficiency after permitting to attach when compared to thosefrom spontaneous differentiation without treatment (control), asassessed by the percentages of cellular clusters that displayed rhythmiccontractions, and immunopositive for markers characteristic ofcardiomyocytes, including Nkx2.5 (green) and α-actinin (red) (DAPI isblue).

(c) Electrophysiological profiles of the beating cardiomyocytesconfirmed their contractions to be strong, rhythmic, well-coordinated,and well-entrained, with regular impulses reminiscent of thep-QRS-T-complexes seen from body surface electrodes in clinicalelectrocardiograms (data also recorded in Videos, see reference [4]).

(d) Upon removal of bFGF and after permitting the RA-induced neuroblaststo attach, β-III-tubulin- (red) and Map-2- (green) expressing,neurite-bearing cells and pigmented cells (arrow, typical of those inthe ventral mesencephalon) began to appear with a drastic increase inefficiency when compared to similarly cultured cells derived fromembryoid bodies (EBs) without treatment (control).

(e) Such preparations could also be dissociated with trypsin andmaintained as a monolayer wherein the RA-induced cells continued topursue a neuronal fate as suggested by their β-III-tubulin (red) andMap-2 (green) immunopositivity.

(f) Nurr1 translocates to the nucleus upon exposure of hESCs to RA.Nurr1, a member of the orphan nuclear hormone receptor super-family, hasbeen implicated in neuronal development, particularly ventralmesencephalic development and activation of the tyrosine hydroxylase(TH) gene, the rate-limiting step in catecholaminergic and dopaminergicneuronal differentiation. In undifferentiated hESCs, Nurr1 localizes tothe cell-surface and cytoplasm, consistent with its being inactive.However, upon exposure of the hESCs to RA, Nurr1 translocated to thenucleus, coincident with the appearance of the neuroectodermal cells,and continued to assume its strong expression and nuclear localizationat the later neuronal stages. All cells are indicated by DAPI stainingof their nuclei (blue).

(g) A large subpopulation of these hESC-derived neuronal cellsprogressed to express tyrosine hydroxylase (TH, red) in the presenceSonic hedgehog (+Shh) or absence Sonic hedgehog (−Shh). Sonic hedgehog(Shh) appeared to promote the proliferation of those ventral neuronalcells. All cells are indicated by DAPI staining of their nuclei (blue).

FIG. 4. Comparing Nurr-1 and Nestin expression and cellular localizationpattern in neuroectoderm-derived human neuronal progenitors direct fromthe pluripotent state of hESCs by RA induction (hESC-I hNuPs) to the twoprototypical neuroepithelial-like human neural stem cells (hNSCs) eitherderived from hESCs via conventional multi-lineage differentiation(hESC-D hNSCs) or isolated directly from human fetal CNS (CNS-D hNSCs)as controls.

(a) RA-induced neuroectoderm-derived hESC-I hNuPs do not express Nestin(green), compared to the two prototypical neuroepithelial-likeNestin-positive hNSCs either derived from hESCs or CNS.

(b) RA-induced neuroectoderm-derived hESC-I hNuPs display strongexpression and nuclear localization of Nurr-1 (green, suggesting itsbeing active), compared to CNS-D hNSCs that show moderate expression andnuclear localization of Nurr-1 and hESC-D hNSCs that show cell-surfaceand cytoplasm localization of Nurr-1 (suggesting its being inactive).

FIG. 5. Neuroectoderm-derived human neuronal progenitors direct from thepluripotent state of hESCs by RA treatment (hESC-I hNuPs) have acquiredpotent neurogenic ability in vitro.

(a) RA-induced neuroectoderm-derived nuclear Nurr-1-positive hESC-IhNuPs differentiated towards a neuronal lineage with a drastic increasein efficiency (˜94%) when compared to the yields of neuronsdifferentiated under similar conditions from the two prototypicalneuroepithelial-like Nestin-positive hESC-D hNSCs (˜6%) or CNS-D hNSCs(˜13%) as controls.

(b) Upon removal of bFGF and after permitting to attach, hESC-I hNuPsyielded exclusively neurons that expressed neuronal marker β-III-tubulinand co-expressed Map-2. No other neural lineages, such as glial cells[e.g., GFAP-positive astrocytes and MBP-positive oligodendrocytes], ornon-neural cells were observed. hESC-I hNuPs yielded neurons efficientlyand exclusively, as they did not differentiate into glial cells,suggesting that these nuclear Nurr-1 positive hESC-I hNuPs are a novelmore lineage-specific neuronal progenitor than the prototypicalneuroepithelial-like Nestin-positive hNSCs.

(c) When dissociated and maintained as a monolayer, the RA-induced cellscontinued to pursue a neuronal fate.

(d, e) Accordingly, a large proportion of these RA-treated hESC-derivedneuronal cells began to express markers associated withventrally-located neuronal populations, such as TH (the tyrosinehydroxylase, marker for dopaminergic [DA] neurons) and Hb9/Lim3/Isl1(markers for motor neurons) (shown in a 3D matrix). All cells areindicated by DAPI staining of their nuclei (blue).

FIG. 6. Genome-scale microRNA (miRNA) profiling of hESC cardiac andneural induction by small molecules.

(a) Hierarchal clustering of differentially expressed miRNAs inundifferentiated hESCs (hESC), cardiac-induced hESCs by NAM (Cardiac),and neural-induced hESCs by RA (Neural) by human miRNA microarrayanalysis.

(b) Pie charts showing decreased contributions of a set ofhESC-associated miRNAs (purple) and increased contributions of distinctsets of cardiac- (green) and neural- (blue) driving miRNAs to the entiremiRNA populations upon cardiac (with NAM) and neural (with RA) inductionof pluripotent hESCs, including silencing of pluripotence-associatedhsa-miR-302 family and a drastic expression increase of neuroectodermalHox miRNA hsa-miR-10 family upon RA exposure.

FIG. 7. Down-regulation of a unique set of hESC-associated miRNAs uponlineage induction by small molecules.

(a) The expression of two most prominent clusters ofpluripotence-associated miRNAs hsa-miR-302 and hsa-miR-371/372/373 wassignificantly suppressed upon lineage-induction of hESCs by smallmolecules. The cluster of hsa-miR-302 family, which had a profile of thehighest expression in pluripotent hESCs, was completely silenced uponneural induction by RA.

(b) A novel group of abundant miRNA clusters in undifferentiated hESCs,including hsa-miR-1308, 3178, 4298, 3195, 1280, 3141, 221/221, and 720,was found to be significantly down-regulated upon small-molecule-inducedlineage differentiation, albeit to less extents. Several clusters ofmiRNAs that were expressed at low levels but share similar or identicalseed sequences with the hsa-miR-302 cluster, including hsa-miR-517, 518,520, 525, and 367, were also significantly down-regulated upon lineageinduction. The clusters of hsa-miR-17 and hsa-miR-20, which werestrongly expressed in undifferentiated hESCs and which havenear-identical seed sequences with hsa-miR-302 family that have beenimplicated in cell proliferation, were found to be significantlydown-regulated upon RA-induced neural differentiation but not uponNAM-induced cardiac differentiation. In most cases, higher degrees ofdown-regulation of hESC-associated miRNAs were observed in RA-inducedneural differentiation in comparison with NAM-induced cardiacdifferentiation. *: 5-10 fold, **: 10-200 fold, and ***: 200-1000 foldof decrease of expression (green lines: cardiac induction by NAM, bluelines: neural induction by RA).

FIG. 8. Up-regulation of a novel set of cardiac-driving miRNAs uponcardiac induction of hESCs by NAM.

(a) Hierarchal clustering of differentially expressed miRNAs inundifferentiated hESCs (hESC) and cardiac-induced hESCs by NAM(Cardiac).

(b) A group of cardiac-specific miRNAs displayed an expression patternof up-regulation upon cardiac induction by NAM and down-regulation uponneural induction by RA. Among this group of cardiac-driving miRNAs, theclusters of hsa-miR-1268, 574-5p, and 92 family contribute to thehighest increased expression profile in NAM-induced cardiacdifferentiation.

(c) A group of cardiac-specific miRNAs had an expression pattern ofup-regulation upon cardiac induction by NAM but was not significantlyaffected upon neural induction by RA. Among this group ofcardiac-driving miRNAs, the clusters of hsa-miR-320 family, 1975, 1979,103, and 107 contribute to the highest increased expression profile inNAM-induced cardiac differentiation.

These data suggested that a novel set of miRNAs, many of which were notpreviously linked to cardiac development and function, contributes toinitiate the cardiac fate switch of pluripotent hESCs.

FIG. 9. Up-regulation of a novel set of neural-driving miRNAs uponneural induction by RA.

(a) Hierarchal clustering of differentially expressed miRNAs inundifferentiated hESCs (hESC) and neural-induced hESCs by RA (Neural).

(b) A group of neural-specific miRNAs displayed an expression pattern ofup-regulation upon neural induction by RA and down-regulation uponcardiac induction by NAM. Among this group of neural-driving miRNAs, theclusters of hsa-miR-10 family, let-7 family (let-7a, c, d, e, f, g), 21,100, 125b, 23 family, and 4324 contribute to the highest increasedexpression profile in RA-induced neural differentiation. Notably, theexpression of hsa-miR-10 family was silenced in undifferentiated hESCsand displayed a drastic increase (˜95-fold) upon RA-induced neuralinduction. The miR-10 genes locate within the Hox clusters ofdevelopmental regulators, and coexpress with a set of Hox genes torepress the translation of Hox transcripts. The drastic expressionincrease of hsa-miR-10 upon exposure of hESCs to RA suggested that RAmight induce the expression of Hox genes and co-expression of Hox miRNAhsa-miR-10 to silence pluripotence-associated genes and miRNAhsa-miR-302 to drive a neural fate switch of pluripotent hESCs,consistent with our observation of a neuroectodermal phenotype ofRA-treated hESCs. The let-7 miRNAs silence the ESC self-renewal programin vivo and in culture, down-regulating pluripotence factors such as Mycand Lin28.

(c) A group of neural-specific miRNAs had an expression pattern ofup-regulation upon neural induction by RA but was not significantlyaffected upon cardiac induction by NAM. Among the this group ofneural-driving miRNAs, the clusters of hsa-miR-181 family, 9, 125a-5p,99 family, 26 family, 30b, and 335 contribute to the highest increasedexpression profile in RA-induced neural differentiation.

These data suggested that a distinct set of miRNAs, many of which werenot previously linked to neural development and function, contributes toinitiate the neural fate switch of pluripotent hESCs. * 5-10 fold, **10-50 fold, and *** 50-200 fold of increase of expression.

FIG. 10. Genome-scale microRNA (miRNA) profiling of hESC neuronalprogression induced by RA. MiRNA (MiR) signatures of human neuronalprogenitor cells (Xcel-hNuP or hESC-I hNuP) and neuronal cells (Xcel-hNuor hESC-I hNu) derived from hESCs by our novel small molecule inductionapproach. Right Panel: Hierarchal clustering of differentially expressedmiRNAs generated by genome-scale profiling of miRNA differentialexpression in hESCs neuronal lineage-specific progression. Left Panel:Pie charts showing decreased contribution of a set ofpluripotence-associated miRNAs (purple) and increased contribution ofdistinct sets of neuronal progenitor-associated miRNAs (blue) andneuron-associated miRNAs (cyan) to the entire miRNA populations duringhESC neuronal lineage-specific progression. Please note that theexpression of pluripotence-associated hsa-miR-302 clusters (dark purple)was silenced and the expression of Hox miRNA hsa-miR-10 cluster (darkblue) was induced to high levels in these in vitro neuroectoderm-derivedhuman neuronal progenitors and neurons.

FIG. 11. RA-induced hESC-derived human neuronal progenitors (hESC-IhNuPs) are highly neurogenic in the brain following transplantation.hESC-I hNuPs were injected into the cerebral ventricles of newborn miceaffording excellent access to the subventricular zone (SVZ), a secondarygerminal zone from which cells widely migrate. Histological analysis oftransplanted mice at least 3 months post-grafting showed well-dispersedand well-integrated human neurons exclusively at a high prevalence,indicated by anti-human mitochondrial antibody (hMit) (red) and theirimmunoreactivity to Map-2 (green), including Nurr1-positive (green)dopaminergic (DA) neurons, within neurogenic regions of the brain. DAPInuclear marker (blue) stains all cells in the field. No tumors ornon-neuronal cell types were seen. Transplanted mice show hyperactivity, such as fast speed movement, fast spin.

DETAILED DESCRIPTION OF THE INVENTION

Pluripotent human embryonic stem cells (hESCs) hold great promise forrestoring cell, tissue, and organ function. However, realizing thedevelopmental and therapeutic potential of hESCs has been hindered bythe inefficiency and instability of generating desired cell types frompluripotent cells through multi-lineage differentiation. This instantinvention is based on the discovery that pluripotent hESCs maintainedunder the defined culture conditions (i.e., feeder-, serum-, andconditioned-medium-free) can be uniformly converted into a neurallineage or a cardiac lineage by simple provision of small molecules(FIGS. 1-11) [1, 2, 4-13]. In particular, retinoic acid (RA) wasidentified sufficient to induce the specification of neuroectodermdirect from the pluripotent state of hESCs in a defined platform bypromoting nuclear translocation of the neuronal-specific transcriptionfactor Nurr-1 and trigger the progression to human neuronal progenitorsand human neurons of the developing CNS in high efficiency, purity, andneuronal lineage specificity [1, 2, 6-13]. Similarly, nicotinamide (NAM)was identified sufficient to induce the specification of cardiomesodermdirect from the pluripotent state of hESCs in a defined platform bypromoting the expression of the earliest cardiac-specific transcriptionfactor Csx/Nkx2.5 and trigger the progression to cardiac precursors andbeating cardiomyocytes in high efficiency, purity, and cardiac lineagespecificity [1, 2, 4-6, 8]. This invention not only provides a largesupply of clinical-suitable human neuronal therapeutic products forneuron regeneration and replacement therapy against a wide range ofneurological disorders and a large supply of clinical-suitable humancardiac therapeutic products for myocardium regeneration and replacementtherapy against heart disease and failure, but also offers means forsmall-molecule-mediated direct control and modulation of the pluripotentfate of hESCs to a specific lineage when deriving an unlimited supply ofclinically-relevant lineages for regenerative medicine.

The hESCs were initially derived and maintained in co-culture withgrowth-arrested mouse embryonic fibroblasts (MEFs) that compromise thetherapeutic potential of these cells because of the risk of transmittingpathogens, altering genetic background, and promoting the expression ofimmunogenic proteins [1]. Although several human feeder, feeder-free,and artificially-formulated defined culture systems have been suggestedfor hESCs, the elements for sustaining undifferentiated growth remainunsolved [1]. These exogenous feeder cells and molecules help maintainthe long-term growth of undifferentiated hESCs while mask their abilityto respond to differentiation inducing signals/molecules. Therefore,previously, I sought to systematically reduce the needs for the growthof undifferentiated hESCs to minimal essential defined components andidentified bFGF, insulin, ascorbic acid, and laminin as the minimalessential components for sustaining the epiblast pluripotence of hESCsin a defined culture system, serving as a platform for de novoderivation of clinically-suitable hESCs and effectively directing suchhESCs uniformly towards functional lineages with small moleculeinduction [see US Patent Application Documents US20050233446,US20070010011, US20080241919, and PCT Patent Application DocumentWO/2005/065354 for inventions by Parsons, Xuejun Huang].

In order to achieve uniformly conversion of pluripotent hESCs to alineage-specific fate, I have used the defined culture system to screenthe differentiation inducing effect of a variety of small molecules andgrowth factors on the pluripotent state of hESCs [1, 4, 5, 7]. Althoughneural lineages appear at a relatively early stage in hESCdifferentiation, treating hESC-differentiated EBs with retinoic acid(RA) only slightly increased the low yield of neurons [1]. RA was notsufficient to induce the neuronal differentiation of undifferentiatedhESCs maintained under previously-reported conditions containing feedercells or feeder-cell-conditioned media [1]. However, I found that suchdefined conditions rendered small molecule RA sufficient to induce thespecification of neuroectoderm direct from the pluripotent state ofhESCs that further progressed to human neuronal progenitors and neuronsin the developing CNS with high efficiency by promoting nucleartranslocation of the neuronal specific transcription factor Nurr1. amember of the orphan nuclear hormone receptor super-family implicated inventral neuronal development, particularly ventral mesencephalicdevelopment and activation of the tyrosine hydroxylase (TH) gene, therate-limiting step in dopaminergic (DA) neuronal differentiation [1,7-13] (FIGS. 1-5). Similarly, the defined platform renders NAMsufficient to induce the specification of cardiomesoderm direct from thepluripotent state of hESCs by promoting the expression of the earliestcardiac-specific transcription factor Csx/Nkx2.5 and triggeringprogression to cardiac precursors and beating cardiomyocytes efficiently[1, 4, 5, 8] (FIGS. 1-3). This compound was not sufficient when appliedto hESCs-aggregated embryoid bodies (EBs) or hESCs maintained underpreviously-reported conditions containing feeder cells orfeeder-cell-conditioned media [1, 4, 7]. This instant invention providesa system for a well-controlled efficient approach to specify pluripotenthuman cells differentiation exclusively to a clinically-relevant lineageby small molecule induction (as illustrated in FIG. 1).

As illustrated in FIG. 1, upon exposure of undifferentiated hESCsmaintained in the defined culture to RA (10 μM), all the cells withinthe colony underwent morphology changes to large differentiated cellsthat ceased expressing pluripotence-associated markers (e.g., Oct-4) andbegan expressing neuroectoderm-associated markers (e.g., HNK1, AP2, andTrkC) (Stage 1—Human Neuroectodermal Cells) [1, 7] (FIG. 1; 2 a, c).These large differentiated cells continued to multiply and the coloniesincreased in size, proceeding spontaneously to express the earlyneuronal marker β-III-tubulin, but not markers associated with otherlineages, including Pdxl, AFP, and insulin [endoderm], Nkx2.5[mesoderm], and GFAP [glial cells] (FIG. 2c ). The more mature neuronalmarker Map-2 began to appear in areas of the colonies where cells hadpiled up (FIG. 2c ). These differentiating hESCs then formed neuroblaststhat uniformly positive for β-III-tubulin in suspension (Stage 2—HumanNeuronal Progenitor Cells [hESC-I hNuPs]) [1, 7] (FIGS. 1; 3 a). Uponremoval of bFGF and after permitting the neuroblasts to attach,β-III-tubulin- and Map-2-expressing, exuberantly neurite-bearing cellsand pigmented cells (typical of those in the CNS) began to appear with adrastic increase in efficiency when compared to similarly cultured cellsderived from untreated embryoid bodies (EBs) as control (Stage 3—HumanNeuronal Cells in the developing CNS) [1, 7] (FIGS. 1; 3 d; 5). Suchpreparations could also be dissociated with trypsin and maintained as amonolayer wherein the RA-induced cells continued to pursue a neuronalfate as suggested by their β-III-tubulin and Map-2 immunopositivity(FIG. 3e ) and the absence of markers associated with other neural cellssuch as glial lineage, as indicated by no cell expressing GFAP and MBP[1, 7]. Nurr1, a member of the orphan nuclear hormone receptorsuper-family, has been implicated in neuronal development, particularlyventral mesencephalic development and activation of the tyrosinehydroxylase (TH) gene, the rate-limiting step in catecholaminergic anddopaminergic neuronal differentiation. Interestingly, inundifferentiated hESCs, Nurr1 localizes to the cell-surface andcytoplasm, consistent with its being inactive (FIG. 30. However, uponexposure of the hESCs to RA, Nurr1 translocated to the nucleus,coincident with the appearance of the neuroectodermal cells, andcontinued to assume its strong expression and nuclear localization atthe later process-bearing neuronal stages (FIG. 30. Accordingly, a largeproportion of these hESC-derived neuronal cells began to express TH(FIGS. 3g ; 5 d), consistent with the early stages of acquiringcatecholaminergic or dopaminergic potential. Similarly, a proportion ofMap-2+ cells began to express Hb9 and Lim3 (FIG. 5e ), markersimplicated in the early stages of motor neuron development, anotherventrally-located neuronal population. Sonic hedgehog (Shh) appeared topromote the proliferation of those ventral neuronal cells (FIG. 3g ).

As illustrated in FIG. 1, upon exposure of undifferentiated hESCsmaintained in the defined culture to NAM (10 mM), all the cells withinthe colony underwent morphology changes to large differentiated cellsthat down-regulated the expression of pluripotence-associated markers(e.g., Oct-4) and began expressing the earliest marker for heartprecursor, Csx/Nkx2.5, but not markers associated with other lineages,including Pdxl and AFP [endoderm] and Map-2, GFAP, Pax6, and HNK1[ectoderm] (Stage 1—Human Cardiomesodermal Cells) [1, 4, 5] (FIGS. 1; 2a, b). Increased intensity of Nkx2.5 was usually observed in areas ofthe colonies where cells began to pile up (FIG. 2b ). Thesedifferentiating hESCs then formed cardioblasts that uniformly expressedNkx2.5 in suspension (Stage 2—Human Cardiac Precursor Cells) [1, 4, 5](FIGS. 1; 3 a). After permitting the cardioblasts to attach and furthertreating them with NAM, beating cardiomyocytes began to appear afterwithdrawal of NAM with a drastic increase in efficiency (Stage 3—HumanCardiomyocytes and Cardiovascular Cells) [1, 4, 5] (FIGS. 1, 3 b). Cellswithin the beating cardiospheres expressed markers characteristic ofcardiomyocytes [1, 4, 5] (FIG. 3b ). The cardiomyocytes can retain theirstrong contractility for over 3 months. Electrical profiles of thecardiomyocytes confirmed their contractions to be strong rhythmicimpulses reminiscent of the p-QRS-T-complexes seen from body surfaceelectrodes in clinical electrocardiograms [4] (FIG. 3c ). Cardiacspecific transcription factor (Csx) Nkx2.5 is an evolutionally conservedhomeobox transcription factor indispensable for normal cardiacdevelopment. The onset and pattern of early Nkx2.5 expression roughlycoincide with the timing and area of cardiac specification, and Nkx2.5gene continues to be expressed through development in the heart [1].Expression of Nkx2.5 is the earliest marker for heart precursor cells inall vertebrates so far examined and is essential for proper cardiacseptation and formation/maturation of electrical conduction system [1].

Unlike the two prototypical neuroepithelial-like Nestin-positive humanneural stem cells (hNSCs) either derived from hESCs or CNS, this novelhuman neuronal progenitors (hESC-I hNuPs), which have acquired aneuroectodermal identity through RA induction of pluripotent hESCs invitro [1, 7], do not express Nestin, but assume uniformly strongexpression and nuclear localization of Nurr-1 [9] (FIGS. 3f ; 4).Although CNS-D hNSCs, which have acquired their neurectodermal identitythrough in vivo developmental processes, show moderate expression andnuclear localization of Nurr-1, in hESC-D hNSCs, Nurr1 localizes to thecell-surface and cytoplasm, suggesting its being inactive [9] (FIG. 4).Upon removal of bFGF and after permitting to attach, hESC-I hNuPsyielded exclusively neurons that expressed neuronal marker β-III-tubulinand co-expressed Map-2 with a drastic increase in efficiency (˜94%) whencompared to the yields of β-III-tubulin-positive neurons differentiatedunder similar conditions from hESC-D hNSCs (˜6%) or CNS-D hNSCs (˜13%)[9] (FIG. 5a, b ). No other neural lineages, such as glial cells [e.g.,GFAP-positive astrocytes and MBP-positive oligodendrocytes], ornon-neural cells were observed [1, 7]. Such neuronal cell preparationscould also be dissociated with trypsin and maintained as a monolayer(FIGS. 3e ; 5 c). Accordingly, a large proportion of these hESC-derivedneuronal cells began to express markers associated withventrally-located neuronal populations, such as TH (DA neurons) andHb9/Lim3/Isl1 (motor neurons) [1, 7, 9] (FIGS. 3g ; 5 d, e).

Under protocols presently employed in the field, hESC-derived cellularproducts consist of a heterogeneous population of mixed cell types,including fully differentiated cells, high levels of various degrees ofpartially differentiated or uncommitted cells, and low levels ofundifferentiated hESCs, posing a constant safety concern whenadministered to humans. In contrast, hESC-I hNuPs consist of ahomogeneous population of human neuronal progenitor cells with potentialto yield high levels of neuronal cells (˜94%). Accessory cells (e.g.,other neural cells) and inappropriate cells (e.g., undifferentiatedhESCs, cytotoxic cells, and non-neural cells) are undetectable in thenovel hESC-derived cellular product. hESC-I hNuPs yielded neuronsefficiently and exclusively, as they did not differentiate into glialcells, suggesting that these Nurr-1 positive hESC-I hNuPs are a novelmore lineage-specific neuronal progenitor than the prototypicalneuroepithelial-like Nestin positive hNSCs. The small molecule directinduction protocol yields nuclear Nurr-1 positive human neuronalprogenitors and neurons of the developing CNS direct form thepluripotent state of hESCs in high efficiency, purity, andneuronal-lineage specificity, therefore, may minimize the risks ofteratoma and ectopic tissue formation by eliminating the presence ofundifferentiated hESCs and non-neural inappropriate cell types. Thisinvention will dramatically increase the clinical efficacy forgraft-dependent neuron replacement/regeneration and safety ofhESC-derived cellular products for CNS repair. Similarly, the smallmolecule direct induction protocol yields human cardiac precursors andcardiomyocytes in high efficiency, purity, and cardiac-lineagespecificity, therefore, may minimize the risks of teratoma and ectopictissue formation by eliminating the presence of undifferentiated hESCsand non-cardiac inappropriate cell types. This invention willdramatically increase the clinical efficacy for graft-dependentmyocardium replacement/regeneration and safety of hESC-derived cellularproducts for cardiovascular repair.

MicroRNAs (miRNAs) are emerging as important regulators of stem cellpluripotence and differentiation [8, 10]. MiRNAs are small,evolutionarily conserved non-coding RNAs that modulate gene expressionby inhibiting mRNA translation and promoting mRNA degradation. MiRNAsact as the governors of gene expression networks, thereby modify complexcellular phenotypes in development or disorders. miRNA microarrayprofile analysis showed that the expression of two most prominentclusters of pluripotence-associated miRNAs hsa-miR-302 family andhsa-miR-371/372/373 was drastically down-regulated upon lineageinduction by small molecules [8, 10] (FIGS. 6; 7 a; 10). The cluster ofhsa-miR-302 family, which had a profile of the highest expression inpluripotent hESCs, was completely silenced in hESC-I hNuPs (average˜550-fold of decrease) [8, 10] (FIGS. 6; 7 a; 10), suggesting thathESC-I hNuPs, unlike previous hESC-derived cellular products throughmulti-lineage differentiation, do not contain any residual pluripotentcells. A novel group of abundant miRNA clusters in pluripotent hESCs,including hsa-miR-17, 20, 221/222, 1280, 1308, 3178, 3141, and 4298, wasfound to be significantly down-regulated in hESC-I hNuPs, albeit to aless extent [8, 10] (FIG. 6; 7 b; 10). The sensitivity, specificity,robustness, and precision of assays to characterize hESC-derivedcellular products by employing genomic miRNA profiling are sufficient toprovide a reasonable assurance of homogeneity and identity ofhESC-derived cellular products, therefore, safety and efficacy whenadministered to humans.

A group of miRNAs displayed an expression pattern of up-regulation uponneural induction by RA and down-regulation upon cardiac induction by NAM[8] (FIGS. 6; 9 a, b). Among the first group of neural-driving miRNAs(neural-specific miRNA group 1), the clusters of hsa-miR-10 family,let-7 family (let-7a, c, d, e, f, g), 21, 100, 125b, 23 family, and 4324contribute to the highest increased expression profile in RA-inducedneural differentiation [8] (FIGS. 6; 9 b). Notably, the expression ofhsa-miR-10 family was silenced in undifferentiated hESCs and displayed adrastic increase (average ˜95-fold) in neuroectoderm-induced hESC-IhNuPs [8, 10] (FIGS. 6; 9; 10). The miR-10 genes locate within the Hoxclusters of developmental regulators and coexpressed with a set of Hoxgenes to repress the translation of Hox transcripts [8]. The enhancer ofthe mouse Hoxb-1 gene, which controls the RA response and regulates geneexpression predominantly in neuroectoderm, contains a retinoic acidresponse element (RARE) that is not only involved in the ectopicresponse to RA, but is also essential for establishing the early Hoxb-1expression pattern in embryonic development [8]. The drastic expressionincrease of hsa-miR-10 in hESC-I hNuPs suggested that RA might inducethe expression of Hox genes and co-expression of Hox miRNA hsa-miR-10 tosilence pluripotence-associated genes and hsa-miR-302 to drive aneuroectodermal phenotype and a neuronal fate in hESC-I hNuPs [8, 10].The let-7 miRNAs down-regulate pluripotence-associated genes such as mycand lin28 [8, 10]. These data suggested that hESC-I hNuPs have acquireda neuronal identity by silencing pluripotence-associated miRNAs andinducing high levels of expression of miRNAs linked to regulatingneuronal development and function, consistent with their high neurogenicability. A second group of miRNAs had an expression pattern ofup-regulation upon neural induction but was not significantly affectedupon cardiac induction [8] (FIGS. 6; 9 a, c). Among the second group ofneural-driving miRNAs, the clusters of hsa-miR-181 family, 9, 125a-5p,99 family, 26 family, 30b, and 335 contribute to the highest increasedexpression profile in RA-induced neural differentiation [8] (FIGS. 6; 9c). These data suggested that a distinct set of miRNAs, many of whichwere not previously linked to neural development and function,contribute to initiate the neural fate switch and neuronal progressionof pluripotent hESCs [8, 10] (FIGS. 6; 9; 10).

A group of miRNAs displayed an expression pattern of up-regulation uponcardiac induction by NAM and down-regulation upon neural induction by RA[8] (FIGS. 6; 8 a, b). Among the first group of cardiac-driving miRNAs,the clusters of hsa-miR-1268, 574-5p, and 92 family contribute to thehighest increased expression profile in NAM-induced cardiacdifferentiation [8] (FIGS. 6; 8 b). A second group of miRNAs had anexpression pattern of up-regulation upon cardiac induction but was notsignificantly affected upon neural induction [8] (FIGS. 6; 8 a, c).Among the second group of cardiac-driving miRNAs, the clusters ofhsa-miR-320 family, 1975, 1979, 103, and 107 contribute to the highestincreased expression profile in NAM-induced cardiac differentiation [8](FIGS. 6; 8 c). These data suggested that a novel set of miRNAs, many ofwhich were not previously linked to cardiac development and function,contribute to initiate the cardiac fate switch and cardiac progressionof pluripotent hESCs [8] (FIGS. 6; 8).

The analysis of genome-scale miRNA profiling identified novel sets ofdevelopment-initiating small molecule miRNAs upon small-molecule-inducedcardiac- and neural-specification of hESCs [8] (FIGS. 6-10). A uniqueset of pluripotence-associated miRNAs was down-regulated, while novelsets of distinct cardiac- and neural-driving miRNAs were up-regulatedupon small-molecule-induced lineage-specific differentiation of hESCs,including silencing of pluripotence-associated hsa-miR-302 family and adrastic expression increase of neural-driving Hox miRNA hsa-miR-10family upon RA exposure [8] (FIGS. 6-10). This invention opens a newdimension of small molecule-mediated direct control and modulation ofhESC pluripotent fate when deriving an unlimited supply ofclinically-relevant lineages for regenerative therapies. This inventionenables well-controlled efficient derivation of a large supply of robusthuman stem/progenitor/precursor cells and specialized mature functionalcells from pluripotent hESCs that can be used in the clinical settingfor tissue and organ regeneration and repair.

To address whether this novel human neuronal progenitors hESC-I hNuPscould be safely engrafted in the brain and could migrate and retaintheir neurogenic ability in vivo, hESC-I hNuPs were transplanted intothe cerebral ventricles of newborn mice. This route allows excellentaccess to the subventricular zone (SVZ), a secondary germinal zone fromwhich cells widely migrate and respond to appropriate regionaldevelopmental cues. After at least 3 months post-grafting, the mice weresacrificed and processed for histological and immunocytochemical (ICC)analysis. Transplanted hESC-I hNuPs engrafted and migrated widely andyielded well-dispersed and well-integrated human neurons exclusively ata high prevalence, including nurr1-positive DA neurons, withinneurogenic regions of the brain (FIG. 11), demonstrating their potentialfor neuron regeneration/replacement cell therapy [9, 10]. No graftovergrowth, formation of teratomas or neoplasms, or appearance ofnon-neuronal cell types was observed following engraftment.

The invention enables developing human-pluripotent-stem-cell-derivedtherapeutic products and supplies, including patient-specific humanstem/precursor/progenitor cells, disease-targeted specialized humancells, and cell- or bio-engineered human tissues and replacement organsthat can be used in the clinical setting forrepair/reconstruction/replacement of the damaged human body structureand circuitry, as well as developing technologies and methods of humantissue and organ regeneration, including high throughput and highcontent assays, analytical and manipulation tools, therapeuticstrategies, and tissue and organ engineering approaches.

The methods, compositions, tools, and products described herein arepresently representative of preferred embodiments and are exemplary andare not intended as limitations on the scope of the invention. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any now-existing orlater-developed equivalent of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention as claimed. Thus, it will beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand/or variation of the disclosed elements may be resorted to by thoseskilled in the art, and that such modifications and variations arewithin the scope of the invention as claimed.

CONCLUSION

The present invention provides the techniques for high efficient directconversion of pluripotent human embryonic stem cells (hESCs) uniformlyinto neuronal or cardiac lineage-specific functional cell products bysmall molecule induction for CNS or myocardium regeneration. The claimsof the invention are provided a method of generating a neuronal orcardiomyocyte lineage-specific functional cell product from pluripotenthuman embryonic stem cells by promoting neural or cardiaclineage-specific differentiation of pluripotent hESCs using smallmolecule induction. A distinctly claiming subject matter of theinvention is provided a method of using retinoic acid (RA) to induce thespecification of neuroectoderm direct from the pluripotent state ofhESCs in a defined culture platform and trigger the progression to humanneuronal progenitors and human neurons in high efficiency, purity, andneuronal lineage specificity. Another distinctly claiming subject matterof the invention is provided a method of using nicotinamide (NAM) toinduce the specification of cardiomesoderm direct from the pluripotentstate of hESCs in a defined culture platform and trigger the progressionto cardiac precursors and beating cardiomyocytes in high efficiency,purity, and cardiac lineage specificity.

INDUSTRIAL APPLICABILITY

The invention provides human CNS and heart-related cells useful fortransplantation, research, drug development, tissue and organengineering, tissue and organ regeneration, scale-up production,cell-based therapy, and other purposes.

What is claimed as the invention is:
 1. A method of producing cells ofneuronal lineage directly from pluripotent human stem cells, comprisingsteps of: (i) providing a culture of pluripotent human stem cells in adefined medium comprising bFGF, insulin, ascorbic acid, and activin-A,wherein said defined medium is free of serum, free of feeder cells, andfree of feeder cell conditioned medium; (ii) adding retinoic acid (RA)to the culture, wherein the pluripotent human stem cells are cultured inthe presence of RA for a period of time sufficient to cause Nurr-1 totranslocate to the nucleus and cause the pluripotent human stem cells todirectly differentiate into cells of neuronal lineage comprising atleast 90% of neuroectodermal cells; (iii) further differentiating theneuroectodermal cells into a population of cells comprising at least 90%of neuronal progenitor cells; and (iv) further differentiating theneuronal progenitor cells into a population of cells comprising at least90% of neuronal cells.
 2. The method according to claim 1, wherein saiddefined medium comprises: (a) a basal medium; (b) 20-100 ng/ml bFGF; (c)10-30 microgram/ml insulin; (d) 40-60 microgram/ml ascorbic acid; and(e) 20-100 ng/ml activin-A.
 3. The method according to claim 1, whereinat least 70% of the pluripotent human stem cells from the culture ofstep (i) are positive for at least 4 markers selected from the groupconsisting of alkaline phosphatase, Oct-4, SSEA-4, Tra-1-60, Tra-1-81,acetylated histones, Brg-1, hSNF2H, and microRNA hsa-miR-302.
 4. Themethod according to claim 1, wherein the pluripotent human stem cellscomprise a pluripotent human embryonic stem cell line which (i) isderived from the inner cell mass or epiblast of a human blastocyst; (ii)is capable of proliferation in culture for over one year in a definedmedium comprising bFGF, insulin, ascorbic acid, and activin-A, whereinsaid defined medium is free of serum, free of feeder cells, and free offeeder cell conditioned medium; (iii) maintains a stable karyotype inwhich the chromosomes are euploid through prolonged culture; and (iv)maintains the potential to differentiate to derivatives of endoderm,mesoderm, and ectoderm tissues throughout the culture.
 5. The methodaccording to claim 1, wherein the cells of neuronal lineage comprisehuman neuroectodermal cells, human neural stem cells, human neuronalprogenitor cells, human neural cells, human neuronal cells, humanpigmented neuronal cells, human neurons, human dopaminergic neurons,human motor neurons, human ventral mesencephalon precursors, and humanventral neurons.
 6. The method according to claim 1, wherein saidneuroectodermal cells are positive for at least 4 markers selected fromthe group consisting of Nurr-1, AP2, TrkC, beta-III-Tubulin, SSEA-1,microRNA hsa-miR-10, acetylated histones, Brg-1, and hSNF2H.
 7. Themethod according to claim 1, wherein said neuronal progenitor cells arepositive for at least 3 markers selected from the group consisting ofNurr-1, beta-III-Tubulin, Map-2, microRNA hsa-miR-10, microRNAhsa-miR-9, acetylated histones, Brg-1, and hSNF2H.
 8. The methodaccording to claim 1, wherein said neuronal cells are positive for atleast 3 markers selected from the group consisting of Nurr-1,beta-III-Tubulin, Map-2, NeuN, 70 KDa NF, 160 KDa NF, microRNAhsa-let-7, microRNA hsa-miR-143, microRNA hsa-miR-124, and microRNAhsa-miR-210.
 9. The method according to claim 1, wherein said neuronalcells comprise dopaminergic neurons and motor neurons that are positivefor at least one marker selected from the group consisting of tyrosinehydroxylase, Lmx1, Msx1, Pitx3, HB9, Lim3, and Isl1.
 10. The methodaccording to claim 1, wherein the cells of neuronal lineage are negativefor at least 4 markers selected from the group consisting of Oct-4,SSEA-4, Tra-1-60, Tra-1-81, microRNA hsa-miR-302, Nestin, Sox-2,Musashi, Pdxl, AFP, Nkx2.5, MBP, and GFAP.
 11. A method of neuronal celltherapy, comprising (i) producing the cells of neuronal lineage directlyfrom pluripotent human stem cells according to the method of claim 1;and (ii) administering to a patient in need of such neuronal celltherapy product the cells of neuronal lineage.
 12. The method accordingto claim 11 wherein the patient suffers from a neurological disordercomprising neurodegenerative diseases, spinal cord injury, motor neurondisease, Alzheimer's disease, Parkinson's disease, multiple sclerosis,amyotrophic lateral sclerosis, spinal muscular atrophy, brain injury,stroke, and macular degeneration.
 13. The method according to claim 11,wherein the pluripotent human stem cells comprise a pluripotent humanembryonic stem cell line.
 14. A method of nerve system drug discovery,comprising (i) producing the cells of neuronal lineage directly frompluripotent human stem cells according to the method of claim 1, and(ii) a high throughput method to screen a compound for an effect on suchproduct of neuronal lineage cells.
 15. The method according to claim 14,wherein the pluripotent human stem cells comprise a pluripotent humanembryonic stem cell line.
 16. A method of nerve system tissueengineering, comprising (i) producing the cells of neuronal lineagedirectly from pluripotent human stem cells according to the method ofclaim 1, and (ii) adding such product of neuronal lineage cells to astructural template that comprises extracellular matrix, biomaterialscaffold, whole-heart scaffold, tissue scaffold, organ scaffold, andsynthetic or purified matrix proteins.
 17. The method according to claim16, wherein the pluripotent human stem cells comprise a pluripotenthuman embryonic stem cell line.